1. Field of Invention
The present invention relates to an extract for preventing or treating thrombotic diseases, and more particularly to an extract of leech and earthworm with molecular weight of less than 5,800 daltons, and pharmaceutical composition, processes for preparation and use thereof.
2. Description of Related Arts
As a kind of classic drug of removing blood clot, the Chinese Pharmacopeia records three kinds of leeches—Whitmania pigra Whitman, Hirudo nipponica Whitman and Whitmania ac-ranulata Whitman. Among them, Whitmania pigra Whitman is the most widely-used for drug. The main ingredients of leech are protein, polypeptide, amino acids, nucleic acid, enzyme, saccharide, trace elements and etc. The qualitative and quantitative researches about ingredients in leech have been reported for many times. The main methods comprise thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and electrophoresis method. The main micromolecular ingredients in leech are hypoxanthine, xanthine, uracil, uracil riboside and etc. Both Pyrimidine and Purine are unsaturated heterocyclic compounds containing N atoms.
As Chinese traditional medicine, earthworm in the Chinese Pharmacopeia has effects of clearing away heat, calming, getting through arteries and veins, smoothing breathe and diuretic. There are four kinds of earthworm in the Chinese pharmacopeia. They are Pheretima aspergillum (E. Perrier), Pheretima vulgaris Chen, Pheretima guillelmi (Michaelsen) and Pheretima pectinifera Michaelsen. Among them, Pheretima aspergillum (E. Perrier) also known as Lumbricus is the most widely-used for drug. The main ingredients in earthworm are protein, amino acids, nucleic acid, enzyme, saccharide and etc. The qualitative and quantitative researches about ingredients in earthworm have been reported. The main micromolecular ingredients in earthworm are hypoxanthine and compounds containing N atoms.
Extensive researches of conventional arts indicate that leeches and earthworms definitely contribute to treating thrombotic diseases, and compounds of the polypeptides and the nucleotide fragments isolated from the leeches and the earthworms are well anticoagulant and antithrombotic. A U.S. Pat. No. 5,583,111A, issued on Dec. 10, 1996, discloses anti-thrombotic polypeptides for treatment of thrombotic diseases, which have molecular weights of 14 kilo daltons, 9 kilo daltons and 3 kilo daltons and are isolated from extracts of leeches. A patent of JP1996041098A, issued on Feb. 13, 1996, discloses anti-thrombotic materials of polypeptides and nucleotide fragments obtained from leech extracts. A U.S. Pat. No. 4,668,662A, issued on May 26, 1987, discloses polypeptides extracted and isolated from leeches as an effective anticoagulant in the treatment of the thrombotic diseases. As disclosed from Arch. Pharm. Res., Vol. 20, No. 1, pp 17-23, 1977, powder of earthworms is antithrombotic and anticoagulant and has fibrin-like activity.
A patent of US20050032745A1, issued on Feb. 10, 2005, discloses modified low molecular weight heparin (MLMWH) produced from low molecular weight heparin (LMWH) and having a molecular weight of about 5,000 daltons to 9,000 daltons, preferably 5.400 daltons to 8,000 daltons, and more preferably 5,800 daltons to 7,000 daltons, for the treatment of the thrombotic diseases.
Safety is one of key requirements for injection drugs. Biological macromolecules like polypeptides in animal medicine are always both effective ingredients and harmful ingredients causing a safety problem. Materials possessing immunogenicity often are macromolecule, and the larger molecular weight, the better the immunogenicity. Polypeptides with micromolecule often have no or few immunogenicity. Antigens often are macromolecule because the following probably reasons: (1) The relative molecular weight of antigens are bigger, the superficial specific chemical genes are more, so that the effect of stimulating immunologic cells is better and the immunologic response is stronger; (2) Macromolecular antigens having complex chemical compositions and stable structures can not be destroyed and eliminated easily and can stay in human being's bodies for long time. So they can stimulate immunologic system persistently and immunologic response emerges. Insulin human composed of 51 amino acids (standard molecular weight is 5,800 daltons) is the boundary of distinguishing high molecular weight materials with low molecular weight materials. The high molecular weight materials are controlled strictly in injection.
Extracts of leech and earthworm is the active components in Shuxuetong Injection which possesses high performance and immediate effect in treating cardiovascular and cerebrovascular diseases. Chinese patent applications 03148281.3, 200410101538.8 and 200510000266.7 disclose Shuxuetong Injection and processes for preparation thereof respectively. All content of the applications were referred in the present invention. However, potential hazards exist in injecting and dosing with the injections produced by the conventional arts. For example, when relatively many visible extraneous materials or insoluble microparticals exist and the injections have materials possessing immunogenicity, if the injections are further refined or purified, the anticoagulant and antithrombotic effective ingredients may be eliminated so that drug action may be reduced, while eliminating harmful materials causing untoward reactions including allergy.
Conventional arts only disclose pharmacological actions of the leeches and the earthworms and anticoagulant and antithrombotic active constituent therein. As a kind of animal medicine, the leeches and the earthworms have complex chemical ingredients. The conventional arts fail to provide safe, effective and controllable injections of leeches and earthworms fit for injecting and dosing and processes for preparation thereof, which are applied in treatment of cardiovascular and cerebrovascular diseases, especially in emergency treatment of thromboembolic diseases.